Transcriptome analysis of Lactococcus lactis in coculture with Saccharomyces cerevisiae

Abstract

The study of microbial interactions in mixed cultures remains an important conceptual and methodological challenge for which transcriptome analysis could prove to be the essential method for improving our understanding. However, the use of whole-genome DNA chips is often restricted to the pure culture of the species for which the chips were designed. In this study, massive cross-hybridization was observed between the foreign cDNA and the specific Lactococcus lactis DNA chip. A very simple method is proposed to considerably decrease this nonspecific hybridization, consisting of adding the microbial partner's DNA. A correlation was established between the resulting cross-hybridization and the phylogenetic distance between the microbial partners. The response of L. lactis to the presence of Saccharomyces cerevisiae was analyzed during the exponential growth phase in fermentors under defined growth conditions. Although no differences between growth kinetics were observed for the pure and the mixed cultures of L. lactis, the mRNA levels of 158 genes were significantly modified. More particularly, a strong reorientation of pyrimidine metabolism was observed when L. lactis was grown in mixed cultures. These changes in transcript abundance were demonstrated to be regulated by the ethanol produced by the yeast and were confirmed by an independent method (quantitative reverse transcription-PCR).

FIG. 1.

Effect of the addition of unlabeled genomic DNA from partner species on the percentages of labeled cDNA cross-hybridizing spots, by L. lactis IL-1403 DNA microarrays, relative to the phylogenetic distances between L. lactis and the partner species.

FIG. 2.

Evolution of biomass (g·liter⁻¹) substrates and fermentation product concentrations (mM) during the first 8 h of the pure culture of L. lactis IL-1403 (A), the pure culture of S. cerevisiae CEN-PK905 (B), and the mixed culture (C).

FIG. 3.

Evolution of extracellular adenine (A), adenosine (B), hypoxanthine (C), xanthine (D), uracil (E), and uridine (F) concentrations during the first 5 h of pure L. lactis IL-1403 (▴) or S. cerevisiae CEN-PK905 (○) cultures and of the mixed culture (□). The limit of detection is 1 μM.

FIG. 4.

Simplified representation of the pyrimidine nucleotide metabolism in Lactococcus lactis. Only reactions relevant to this study are included in the figure. Genes whose mRNA abundances differed between pure and mixed culture are indicated. Dotted arrows indicate a lower mRNA abundance, while bold arrows indicate a higher mRNA abundance of genes in mixed culture than in pure culture. PRPP, phosphoribosyl-pyrophosphate.

FIG. 5.

Comparison of transcript level ratios of pyrimidine de novo synthesis genes measured with real-time PCR after ethanol or carbonate pulses or with biochips for the comparison between mixed L. lactis/S. cerevisiae cultures and pure L. lactis cultures. Asterisks indicate P values of <0.05 in biochip experiments.